Ocimum basilicum ( kemangi ) intervention on powder and microencapsulated Spirulina platensis and its bioactive molecules

Background: Spirulina platensis contains several bioactive molecules such as phenol, flavonoid and phycocyanin pigments. This study unveils total phenol, flavonoid, antioxidant activity, phycocyanin content and evaluated encapsulation efficiency from Ocimum basilicum intervention on S. platensis. O. basilicum intervention aims to reduce unpleasant odors from S. platensis that will increase consumption and increase bioactive compounds. Methods: The intervention was carried out by soaking a S. platensis control sample (SP) in O. basilicum with a ratio of 1:4 (w/v) and it was then dried (DSB) and microencapsulated by freeze drying methods (MSB) using a combination of maltodextrin and gelatin. Total flavonoid and phenolic analysis with curve fitting analysis used a linear regression approach. Antioxidant activity of samples was analysed with the 2,2’-azino-bis-3-3thylbenzthiazoline-6-sulphonic acid (ABTS) method. Data were analysed using ANOVA at significance level (p < 0.05) followed by Tukey test models using SPSS v.22. Results: The result of this study indicated that O. basilicum intervention treatment (DSB) has the potential to increase bioactive compounds such as total phenol, antioxidant activity and phycocyanin, and flavonoid content. Intervention of O. basilicum on S. platensis (DSB) significantly increases total phenol by 49.5% and phycocyanin by 40.7%. This is due to the phenol and azulene compounds in O. basilicum which have a synergistic effect on phenol Open Peer Review


Introduction
Spirulina platensis is a blue-green microalga that thrives in alkaline water and it has a high potential as a source of bioactive compounds with commercial importance 1,2 . High value compounds with interesting functional properties such as phycobiliproteins consisting of phycocyanins and allophycocyanins, carotenoids, phenolic acids, omega-3 and omega-6 polyunsaturated fatty acids, phenol and flavonoid have been identified in S. platensis [3][4][5] . Phenolic compounds are a source of bioactive molecules with several beneficial health effects 6 due to their ability to act as antioxidants 7 , antibacterial 8 , and antidiabetes agents 9 . Phycobiliproteins, carotenoids and phenol present in S. platensis have anti-inflamatory activities 10 , thus making them a potential functional food product 11 .
Ocimum basilicum, commonly know as sweet basil or kemangi in Indonesia and called rehan in Arabic 12 is a popular culinary herb. O. basilicum is added to a variety of foods to impart a specific aroma. O. basilicum contains essential oils such as chavicol, linalool and eugenol, which are widely used in the food and pharmaceuticals industries 13 . The essential oils are able to reduce unpleasant odors and replace antioxidants 14,15 . Besides essential oils, basil also contains phenol and flavonoid compounds which have antioxidant properties [16][17][18] .
Microencapsulation is a technique used to coat a material to protect the material from outside factors, as well as ease handling of the material. The most important factor in encapsulation is the type of coating used. The encapsulated material is referred to as the core, intenal phase-, or filler, whereas the walls are sometimes called shells, layers, material wall, or membranes. A microcapsule can be coated by several coatings, but only one core compound can be coated 19,20 .
To predict the potential for bioactivity, absorption, distribution, metabolism, and excretion of a substance, our research was performed with bioinformatics and in silico approaches. If we do not have special apps, certain internet-based or online resources can be used. DOCK Blaster for molecular docking prediction 21 , MDWeb and MDMoby for molecular dynamics analysis 22 , ADMET and DrugBank for drug database creation 23 , as well as PreADME for ADMET tools 24 , are some of the tools available online.
Various studies have reported the presence of bioactive compounds such as phenols and flavonoid in S. platensis 25 and O. basilicum 26 . The present research aims to evaluate bioactive compounds of O. basilicum intervention on S. platensis. Firstly, total phenol, flavonoid, antioxidant activity and phycocyanin contents were evaluated. Secondly, the success of encapsulation of phenol, flavonoid, antioxidant activity and phycocyanin compounds was evaluated. The addition of these compounds was expected to reduce the amount of volatiles in S. platensis, which cause unpleasant odors. The third was predicting absorption, distribution, metabolism, and excretion (ADME) of phenols, azulene, flavonoids, and phycocyanin.

Methods
S. platensis powder was obtained from brackish water Aquaculture Fisheries (BBPBAP) Jepara (Central Java, Indonesia), O. basilicum was bought from a traditional market (Semarang, Central Java). The water used was multilevel distilled water, aquabidest Otsu-WI (PT. Otsuka Indonesia, Lawang, Indonesia). The reagents and chemicals used in this study were of analytical grade (CV. Chemix Pratama, Special Region of Yogyakarta, Indonesia), maltodextrin (CV. Multi Kimia Raya, Semarang, Indonesia) and gelatin (Xian, Biof Bio-Technology, Cina). This research was conducted in the food chemistry laboratory of Diponegoro University, Semarang, Central Java, Indonesia) from January 06, 2020 up to May 29, 2020.

Preparation of Ocimum basilicum leaf extract
The O. basilicum was extracted using distilled water (aquabidest) following modified methods reported by Handiani et al. 27 . 2000 g of fresh O. basilicum leaves were added to 400 ml aquabidest and ground. The slurry was then filtered by using filter fabric and the extract result was approximately 1200 ml.
Intervention of Ocimum basilicum on S. platensis A freeze-dried sample (DSB) and microencapsulation sample by freeze drying (MSB) of S. platensis were soaked with O. basilicum extract for 10 min with ratio of 1:4, w/v. A S. platensis sample with no O. basilicum added was used as a control (SP).

Preparation of the intervention of Ocimum basilicum on
Spirulina platensis by freeze drying O. basilicum and S. platensis were freeze-dried using a freeze dryer (Heto Powerdry LL 1500, Germany) at a temperature of -100°C for 48 hours. The O. basilicum extracts were applied to S. platensis (DSB) in the intervention study below.
Microencapsulation of the intervention of Ocimum basilicum on Spirulina platensis by freeze drying This Microencapsulation was performed following the methods reported by Castro-Munoz et al. and Dewi et al. 28,29 Ten percent (10%) of coating materials (64 g) a consisting maltodextrin and gelatin at a ratio of 9:1, w/w were used for microencapsulated. Then, S. platensis were soaked with O. basilicum extract were

Amendments from Version 1
We have added the explanation of the MSB sample method and the encapsulation efficiency analysis procedure. The result of the standard curve has been changed to the equation of the standard curve line which is placed in the method section. SPSS analysis results tables have been deleted. Figure of the results of bioactive compounds (total phenol, flavonoids and antioxidant) have been separated into three images and re-analyzed ANOVA. In the discussion section, organoleptic results and analysis of volatile compounds as well as the synergistic relationship of phycocyanin and phenol with antioxidant activity have been added.
Any further responses from the reviewers can be found at the end of the article REVISED added into the mixture. Homogenization was then performed with a homogenizer (15A HG-wiseTis, Germany). S. platensis treated with microencapsulation freeze-dried O. basilicum (MSB) was used in the intervention studies below.
Determination of total phenol content Total phenol content was measured using modified Folin-Ciocalteu methods 30 . Samples were sonicated for 30 minutes prior to measurement. Gallic acid was used as standard and was read at λ=739 nm using a UV-Vis spectrophotometer. In the test solution, 0.5 ml of Folin-Ciocalteu reagents and 1 ml of NaCO 3 were added to 1 ml of sample and the solution was mixed. Samples were incubated for 10 minutes at room temperature, then diluted with aquabidest to 10 ml. The measurement results were reported in milligram (mg) and were calculated as gallic acids equivalent (GAE) per gram of sample. The result of the gallic acid calibration curve obtained equation y = 1.0677 x -0.0022 with a value R 2 = 0.9915.

Determination of flavonoid content
Measurement of total flavonoid was performed using the slightly modified aluminium chloride method 31 . Modification was through ultrasonic treatment before measurement, the sample was sonicated for 30 minutes and quercentin was used as a standard. In the test solution, 1.0 ml of sample was mixed with 0.3 ml of NaNO 2 (5%, w/v) and the solution was left to stand 5 minutes before 0.5 ml of AlCl 3 (2%, w/v) was added to the test solution. Samples were neutralized with 0.5 ml of 1 M NaOH solution and the samples were incubated for 10 minutes at room temperature. Absorbance was measured at λ=310 nm. The results are presented in milligrams (mg) and calculated as quercentine equivalent (QE) per gram of sample. The result of the quercetin calibration curve obtained equation y = 0.0185 x + 0.0223 with a value R 2 = 0.9995.
Phycocyanin content 40 mg of sample was added into 10 ml centrifugal tube phosphate buffer (pH 7) 100 mM; the solution was sonicated for 30 minutes and stored at 4°C overnight. Samples were centrifuged to separate the blue supernatant. Next, samples were measured for absorbance at 620 nm according to the methods described by Setyoningrum & Nur 32 . Phycocyanin content was determined using Equation 1: Where PC is phycocyanin content, Abs is absorbance at 620 nm; v is volume of solvent (ml); 3.39 is the coefficient of C-Phycocyanin at 620 nm; w is weight of sample (mg); and w dry is percentage dry weight of sample.

Determination of antioxidant activity
The antioxidant activity of the sample was measured by 2,2'-azinobis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical according to the methods of Shalaby & Shanab 33 . ABTS was formed by reacting 7 mM ABTS aqueous solution with 2.45 mM phosphate per sulphate in the dark for 4-16 hours at room temperature. Dilute ABTS solution with ethanol absorbance of 0.700 ± 0.05 at 734 nm was used for measurement. The photometric test was carried out with 0.9 mL ABTS solution and 0.1 mL of the tested sample mixed for 45 seconds, measurements were made immediately at 734 nm after 15 minutes. Antioxidant activity was expressed as the inhibition percentage of free radicals by the sample and was determined using Equation 2: = Ab -As Inhibition (%) 100 Ab Where Ab is the absorbance of the control reaction and As is the absorbance in the presence of the extract sample.

Determination of encapsulation efficiency
Encapsulation efficiency (EE) was determined following the methods described by Ong et al. 34 . Encapsulation efficiency was calculated based on total coated active compounds and free active compounds. Percent encapsulation efficiency was determined using Equation 3: Total coated active compounds Free active compounds (%) 100 Total coated active compounds Where total coated active compounds in the total active compounds such as phycocyanin, phenol, flavonoid and antioxidant in microcapsule (MSB sample). While free active compounds is the mass of active compounds such as phycocyanin, phenol, flavonoid and antioxidant in microcapsule (powder) surface.
Free active compounds mass was calculated as follow: • Phycocyanin (40 mg microcapsule were washed with 10 ml of buffer phosphate) • Total phenol (1 g microcapsule were washed with 9 ml of aquabidest) • Flavonoid (50 mg microcapsule were washed with 5 ml of methanol) • Antioxidant activity (20 mg microcapsule were washed with 2 ml of ethanol) The solution were filtered using Whatman paper No.42. After filtration, the free active compounds was measured according to the same methods described for active compounds such as (phycocyanin, total phenol, flavonoid and antioxidant activity) determination.
Parameter of separation of the free active compound from the encapsulation is the solubility of the active compounds when washed by strirring for one minute.

ADME analysis
The research was performed in two phases, namely: the first stage of accessing the PubChem server (https://pubchem.ncbi. nlm.nih.gov/) to obtain canonical SMILE information; the next step is to use swiss ADME (http://www.swissadme.ch/) to predict absorption, distribution, metabolism, and excretion 35 . The BOILED Egg (Brain Or IntestinaL EstimateD permeation predictive model) methods are used for the determination of the absorption of the inhibitors in the brain and gastrointestinal tract. BOILED Egg provides a threshold (TPSA ≤ 131.6 and WLOGP ≤ 5.88) and the best representation of how far molecular structure is for well-or poorly absorbed 36 . ADME is based on the Lipinski rule of five 37 . The Lipinski rule of five is generally employed in accessing the drug-likeness of active compounds to prioritize compounds with an increased likelihood of high oral absorption 38 .

Statistical analysis
Data obtained was reported as the mean of triplicates (n=3) ± standard deviation. Parametric data was analyzed using SPSS version 22.0 (IBM, Armonk, NY, USA) 39 . Statistical analysis was preceded by a normality test with One Sample Kolmogorov-Smirnov Test and a homogeneity test with the Levenes Test at significance level (P > 0.05). Parametric tests were carried out with One Way ANOVA at significance level (P < 0.05), followed by post hoc Tukey HSD. This is encapsulation is less effective in microencapsulation of polyphenol compounds such as phenol and flavonoid ( Figure 5). Raw absorbance data for bioactive compounds assays are available as underlying data 40 .

Discussion
Microalgae are a valuable source of proteins and phenol compounds. S. platensis is a type of microalgae with a high total phenol content 41 . Extraction methods and the solvent used are responsible for the type and yield of phenolic compounds from algae sources 42 . In S. platensis, distilled water has been reported as the best solvent for extraction of phenolic compounds with total phenol content of 43.2 ± 1 mg GEA/g 43 . S. platensis powder prepared via oven drying is reported to have a broad range phenolic profile that includes gallic acid, catechin, caffeic acid, P-hydroxybenzoic acid, P-cumaric acid, ferulic acid, quercein, genistein and kaempferol 44 . Variation in total phenol content between algae species is reportedly due to algal type, origin and growth condition of different microalgae 45 .
Fresh O. basilicum leaf extract has been reported to have lower total phenol content than that which has been freeze-dried 46   Another study reported that the powder of S. platensis, which was dried in an oven at a temperature of ± 50°C, did not effect the phenolic compound quercentin, where the compound was one of the active substances of the flavonoid class 44 . The flavonoids are considered as indispensable in a variety of medicines, nutraceutical, pharmaceutical and cosmetic applications 51 . Flavonoids derivative compounds play an antioxidant namely hesperidin and quercetin 52 . The optimum for the extraction process are dry conditions compared to wet conditions. Extraction using ethanol had a higher total flavonoid content 53 . The total flavonoid content of the S. platensis microencapsulated and freeze-dried tended to be low. Microencapsulation can maintain the stability of flavonoid from processing effects that cause degradation 54,55 .   pigments such as phycocyanin of S. platensis produce strong antioxidants.
Ocimum basilicum contains 65 active compounds, and the compounds with the highest content are namely 31.6% linalool and 23.8% methylchavicol. Essential oils in O. basilicum have the potential as antioxidants 13 . The essential oil of linalool significantly prevents the formation of UVB-mediated 8-deoxy guanosine, which causes oxidative damage to DNA.  Encapsulation efficiency is used to evaluate the success of a microencapsulation technique. Encapsulation using a combination of polyanion and polycation coatings such as maltodextrin and gelatin has a higher yield. This is due to the stability of the emulsion between maltodextrin and gelatin 73 .
The amount of bioactive content on the surface will reduce the value of encapsulation efficiency. This will cause the amount of bioactive compounds that are wrapped to increasingly shrink because many are attached to the surface. So that this event will damage the oxidative stability of microcapsules 74 . The encapsulation efficiency of phycocyanin was in accordance with the results of previous studies 75 , which is encapsulation using an alginate coating has an encapsulation efficiency value of 71.75%. The value of encapsulation efficiency in total phenols and flavonoids in S. platensis is effected by using liposomes or nanoliposomes in encapsulation of bioactive compounds, this is because liposome is stable at low pH and is able to withstand the time of release in the stomach, but it is less consistent in the intestine 76,77 . The encapsulation efficiency of antioxidant has been shown in previous research where antioxidant microencapsulation using the freeze drying method has an encapsulation efficiency value ranging from 73-86% 78 .
Intestinal absorption and brain permeation set crucial parameters at their target site of action for any medication for its pharmacokinetics and bioavailability. Consequently, the BOILE-DEgg study was used, as previously stated, to predict gastrointestinal (GI) absorption and brain access for phenol, azulene, flavonoid, and phycocyanin. The white region is the physicochemical space of the molecules most likely to be consumed by the gastrointestinal tract, whereas the yellow region (yolk) is the physicochemical space of the molecules most likely to reach the brain. The white and yolk regions are not mutually exclusive 36 . Phenol, azulene, and phycocyanin were found to be among the well-absorbed molecules based on the study (Table 1). Tables 2 and Table 3 demonstrate that phenol, azulene, and phycocyanin comply with Lipinski or drug-likeness laws. Drug-likeness is a term used to explain how in vivo molecular properties are influenced by compounds' physicochemical properties. This research indicates that the substance will spread well to all parts of the body to play an active role as a drug 79 . The physicochemical properties obtained from molecular structures are used by most drug-likeness testing laws and compare such properties with the medicines that have been reported. The Lipinski rule is one of the most used rules 80 . The rule of five was developed to set drugability guidelines for new molecular entities (NMEs) 81 . Therefore, the rule suggests that molecules, whose properties fall outside of these boundaries, are unlikely to become orally bioavailable drugs 82 . As drug candidates, phenol, azulene, and phycocyanin have excellent potential. This calculation is based on a molecular weight (MW) value of less than 500 g mol -1 , an acceptor of hydrogen bonds of less than 10, a donor of hydrogen bonds of less than five, a surface area of topology (TPSA) of less than 140 Å, and a LogP of less than five.

Conclusion
Ocimum basilicum intervention significantly increased total phenol, phycocyanin and antioxidant activity in S. platensis. However, total flavonoid content did not differ significantly in untreated S. platensis controls compared to treated. Bioactive compounds after microencapsulation showed the lowest values. Microencapsulation of phycocyanin with maltodextrin and gelatin showed high encapsulation efficiency values. Hence, S. platensis treated freeze-dried O. basilicum has potential as a functional foods and pharmaceutical product.

Open Peer Review
freeze-dried and microencapsulated Spirulina platensis. In general, I find this manuscript provides an important contribution in developing palatable and higher bioactive compounds in S. platensis. However, I have outlined below a few issues that need to be addressed before publication, and some more minor comments and suggestions to further improve the quality of the manuscript.

Major:
The methods section is not clear. It is not clear whether O. basilicum alone was freeze-dried and/or microencapsulated, or the S. platensis alone, or is it after the intervention of O. basilicum to S. platensis? Example: In the sub-methods title "Intervention of Ocimum basilicum on S. platensis", the authors stated "…freeze-dried sample (DSB) and microencapsulation sample (MSB) of S. platensis…", while in the sub-methods title "Preparation of freeze-dried Ocimum basilicum", the authors described that both O. basilicum and S. platensis were freeze-dried using a freeze dryer, and followed by a description of the "Microencapsulation of freeze-dried Ocimum basilicum". Please clarify and make changes to the methods section as appropriate. 1.
In the abstract and introduction, the authors stated that one of the aims of the studies is to reduce unpleasant odours of S. platensis by intervention with O. basilicum. However, the organoleptic properties were not presented and discussed.

2.
The authors tried to predict the ADME of the bioactive compounds in the product (Ocimum basilicum -Spirulina platensis) by using the SwissADME. However, as this approach can only predict pharmacokinetics, drug-likeness of small molecules, the authors use four main compounds found in the product namely phenol, azulene, flavonoid (not clear on which flavonoids), and phycocyanin (the structure of each compound used for analysis need to be added to Table 7). While this information provides a prediction of an individual molecule of the complex bioactive compounds in Ocimum basilicum -Spirulina platensis, it does not provide any additional insight to the manuscript as matrix-derived combination influences ADME. In vivo study is needed to determine the bioavailability of Ocimum basilicum intervention on freeze-dried and microencapsulated Spirulina platensis. If the authors wish to include this data in the current study, additional discussion in limitations and future studies include the need for in vivo study are needed.

3.
In the method section, it is not clear how the encapsulation efficiency was determined. Do the authors analyze the active compounds in both the free and encapsulated fractions? If yes, please describe the procedure and parameter of separation of the free active compound from the encapsulation.

4.
In the discussion section, it is not clear how this research indicates a synergistic interaction between phycocyanin and total phenols in antioxidant activities (page 9 of 16). No data support this statement.

5.
Figure 4 is data on encapsulation efficiency. In the discussion (Page 9 of 16), by using the information in Figure 4, the authors stated the effect of different cultivation methods of S. platensis on phycocyanin content. No data support this statement.

Minor:
In the result section paragraph 2 (page 4 of 16), please add a description of the result of MSB. 1.
In the method section, microencapsulation, please describe what is the total solid content of coating materials used in the study.

2.
In the discussion section paragraph 3 (page 6 of 16), please be clear on what bioactive compounds are the authors referring to and for which treatment. 3.
In the introduction and abstract, O. basilicum intervention to S. platensis is to increase consumption and increase bioactive compounds and their potential for a functional food product. The conclusion state as a potential for a pharmaceutical product. Please be consistent.

5.
In Figure 2, B = O. basilicum leaf extract was analyzed. Please describe B in the method section.

6.
The paper would benefit from a more detailed discussion regarding the organoleptic properties of how the Ocimum basilicum intervention reduces unpleasant odours as outlined in the comments above.
○ "Gallic acid was used as standard and was read at λ=739 nm using a spectrophotometer"add the type of spectrophotometer. ○ "Algae are a valuable source of proteins and phenol compounds" -A algae means macroalgae, be careful with this statement.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?